DE4217891A1 - Biocatalyst based on polyurethane - Google Patents

Description

The invention relates to a biocatalyst with immobilized Cells or enzymes, in which the cells or enzymes in one Matrix based on a polyurethane with masked Isocyanate groups are immobilized and a process for Production of such a biocatalyst.

Find biocatalysts with immobilized cells or enzymes used in a wide variety of areas. The application is particularly useful in areas where the cell len or enzymes not in the biotransformation Product can or should remain so. B. in alcohol fermentation, in the manufacture of pharmaceuticals or in Wastewater treatment.

Matrices made of polyurethane hydrogels have very good mecha properties, but have the major disadvantage that the urethane prepolymers used to manufacture them due to the free isocyanate groups present a high toxicity cells and enzymes. The high toxicity usually leads to more or less severe damage the cells during immobilization and thereby to an activi loss of activity of those made with these urethane prepolymers Biocatalysts compared to free cells.  

In addition, the processing of the urethane prepolymers by the Kur ze processing time subject to severe restrictions, be due to the rapid crosslinking reactions in the range of Seconds.

JP 205 127 describes a method for immobilizing Mi known microorganisms in which water-soluble urethane prepolymer Bisulfite adducts are used. This urethane prepolymer Bisulfite adducts are made by this process in an aqueous Processed buffer solution with a pH <9. The reaction solution is used to produce a biocatalyst on a membrane sprayed.

However, these membranes have the major disadvantage that they often not due to their low mechanical stability are settable. There is therefore still a need for organic catalysts in three-dimensional, stable form, e.g. B. as Balls or threads.

The problem underlying the invention arises from the fact that only a biocatalyst of the type mentioned is extremely limited use.

Based on this problem, the invention is Characterized biocatalyst of the type mentioned net that the biocatalyst has a three-dimensional shape points by introducing a first solution into a second Solution with immediate networking of the first solution in the second Solution can be produced.

The biocatalyst according to the invention preferably has one Drop or spherical shape on or is as a thread (with or without a mechanical support).  

The biocatalyst according to the invention therefore has one for bio conventional form and is compared to known alginate Mechanically much more stable catalysts. The fiction moderate biocatalyst can not be biodegradable in a Configuration can be created when a PCS is based on a Polyether polyol is used. However, it is a biological one Degradability is desired, this can be done by training the PCS the base of a polyester polyol can be achieved.

Due to the short processing time of the polyurethane Prepolymers not yet possible to produce spherical Biocatalysts succeed with special Ver drive.

In a first of these methods, the cells or enzymes in an aqueous solution of a capped polyurethane pre polymers with a first cross-linking partner, the one with a second networking partner not in the solution rea can yaw, suspended at a pH <7 and so formed dete suspension ent in a second cross-linking partner holding second liquid introduced. At or after training A cross-linked form becomes the pH value within the core raised to <7 to crosslink the polyurethane.

This process is based on the knowledge that the masked Polyurethane prepolymers that are no longer used for cells or enzymes are toxic, with a pH <7 not or only slowly ver network, d. H. remain processable. In this processable Form is created by introducing the suspension into the second liquid form stabilization immediately through a networking craze tion between the two networking partners. These Crosslinking reaction does not yet affect the crosslinking of the bottom lyurethans. Only when stable forms through networking, at balls or threads, for example, are also found the crosslinking of the polyurethane takes place by the Biokataly surrounding pH is raised. This is there regularly by happening that the second liquid has a pH  <7 is set. In this case, care must be taken that the crosslinking reaction between the two crosslinking partners runs faster than the crosslinking of the polyurethane. Introducing the polyurethane prepolymer into a second stream liquid with a pH between 7 and 10 without a shape de Measure would not lead to usable forms of biocatalysts ren.

The preferred material for the processes described is PCS.

The first crosslinking partner can be used for the shaping reaction an ionotropic, gel-forming liquid and as a second ver an ionic crosslinking agent will. A suitable and proven polyelectrolyte for this One case is Na alginate, which can be crosslinked with Ca ions. In this Fall forms a stable catalyst with the suspension pearl out in the polyurethane by exposure to pH can harden, which is set between 7 and 10. The butt lyurethane matrix is then formed between the gelma formed trix that can be preserved. So-called "Inter penetrating networks ".

In an alternative embodiment, the ionic ver wetting agents are added to the suspension and the second Liquid which has a gel-forming liquid. In this Case arises when the suspension is dropped or injected the second liquid is a network from the inside out, So from the suspension into the second liquid, where through the second liquid a shell around the suspension trains. When the suspension is dropped, hollow spheres are formed, the hal the shape of the suspension when curing the polyurethane A cross-linked polyurethane core with a Shell made of alginate, for example. This envelope can then be removed.

Another method is as a networking partner to use two crosslinking monomers, which in  Introduce the suspension into the second liquid a limit cause layer polymerization and thus also a shell form from the polymer formed around the suspension and so be the shape of the suspension during polyurethane curing vote. This shell can also be used after curing the polyurethane be removed ethans.

It is also possible to shape, i. H. for training a stabilizing shell, two polyelectrolytes as a network partner to use. It is in the suspension expediently a quaternary ammonium salt such as. B. Polydime thyldiallylammonium chloride. The second liquid then contains Another polyelectrolyte that is used when introducing the suspension immediately with the polyelectrolyte in the suspension cross-linked, whereby the shape of the catalyst is stabilized. In the case of the polydimethyldiallylainmonium chloride mentioned above can e.g. B. cellulose sulfate used as a crosslinking agent the. The polyurethane is crosslinked after the Formation of the casing also by the action of the pH value, which is preferably set between 7 and 10. By Polyelectrolyte-polyelectrolyte crosslinking is formed like this thin that it does not have to be removed in this process.

An alternative to the shape described so far Networking of two networking partners is given by the fact that the shaping takes place through a suitable temperature selection. Also here the suspension is at a pH <7 at a first temperature kept processable. The first temperature can be room temperature for PCS. The suspension is then in the second liquid is introduced, which is used for curing of the polyurethane required higher pH and a second temperature is set that is higher than that first temperature and at which the polyurethane prepolymer is not is more water-soluble and immediately keeps its shape. In the precipitated phase then cross-links the polyurethane instead of. The second liquid can be a buffer solution. For PCS a suitable second temperature is between 38 and 40 ° C.  

A preferred introduction of the suspension into the second liquid speed is achieved by dropping. This creates for everyone described methods spherical or teardrop shapes for the bioka talysator.

Alternatively, it is possible to use the suspension as a continuous to introduce the liquid jet into the second liquid, whereby a thread-like biocatalyst can be produced.

It is also possible to use the suspension with a central Nozzle to form a drop that coincides with its formation the second liquid is surrounded. This is through one first annular nozzle surrounding annular nozzle possible, ie with the arrangement of a known two-component nozzle.

Furthermore, it is possible with the known two-component nozzle formed as a hollow sphere or hollow fiber biocatalyst put. For this purpose, a cell suspension, which may be a means for Increase in viscosity contains from a central nozzle wear while at the same time reversing the central nozzle the annular nozzle a solution that the PCS prepolymer and e.g. B. contains Ca ions, is discharged simultaneously, so that Drops of liquid emerging from the first central nozzle or jet emerging from the second annular nozzle liquid is enclosed. The networking then takes place in the manner already described by a second liquid.

The particular advantage of this procedure lies in the training a hollow sphere, the gel layer of which is cell-free and in the In an unimpeded mixing of the gel layer diffusing substrate with the suspension is possible.

The PCS prepolymer is at a pH <2 and at a tem temperature below -18 ° C for unlimited storage. It can be for everyone Procedure before processing on one for the cells or En zyme compatible pH from 4 to 6 can be adjusted.  

For processing urethane prepolymers and shaping of the biocatalyst is the gel time and the viscosity of the Prepolymer suspension of particular containing cells or enzymes their meaning. The water solubility of the is also important Prepolymers, so that no cosolvents are used can be.

In line with results published in 1959 (Bailey Jr., F.E., Collard, R.W., J. Appl. Polym. Sci. 1 (1959) No. 1, pp. 59-62) it was found that with increasing poly ethylene oxide content of polyethylene oxide and polypropylene polymer units composed of oxide units here were looking for urethane prepolymers that increase water solubility while rend with increasing molecular weight and with increasing salt concentration in the solution the water solubility expected decreases moderately. Accordingly, in an advantageous design of the method according to the invention urethane prepolymers used that have a polyethylene oxide content of over 70% point.

The viscosity was used to determine the viscosity optimal drip behavior for the production approx spherical biocatalysts is achieved. According to the invention are accordingly in a preferred embodiment prepo lymer concentrations of preferably 10-20% to the above called biocatalyst processed. In particular, be particularly preferred configurations of the method are the concents ratios of prepolymers to 15% at the pH of 4 (PCS 15-4), 12.5% at a pH of 5 (PCS 12.5-5) and to 10% at a pH set from 6 (PCS 10-6).

By using the urethane prepolymer bisulfite adducts, others Designation Polycarbamoylsulfonate (PCS), corresponding to the before lying processes, it is possible to produce biocatalysts len, whose activity is at least comparable to that of biocata analyzers based on biopolymers, such as Ca alginate, potassium  um-karrageenan etc. This beneficial effect is not based only on the capping caused by the reaction with bisulfite of the reactive and toxic isocyanate groups, but also because the capping reaction in a water / isopropanol mixture takes place and the isopropanol largely after the reaction ent can be removed, with low isopropanol back in practice remains in the PCS prepolymer, which are practically none toxic influence on the cells or enzymes to be immobilized me exercise.

In the following, the invention is intended to be used together with examples menhang with the drawings are explained in more detail. Show it:

Fig. 1 is a diagram of cell activity of S. cerevisiae;

Fig. 2 is a graph showing the dependence of the gel time of the pH;

Fig. 3 is a diagram of the optimal dripping behavior at the game of three different PCS solutions;

Fig. 4 is a scheme for the preparation of PCS-spheres;

FIG. 5a is a graph comparing the formation of ethanol by S. cerevisiae in various polymer matrices;

Fig. 5b is a diagram of the relative ethanol formation activity of S. cerevisiae in a Ca-alginate and a polyurethane matrix;

Fig. 6 is a graph comparing the nitrate removal activity of immobilized cells of P. denitrificans in Ma trizes different based on NCO prepolymers;

Figure 7a is a diagram of the ammonium-free and degradation with immo-stabilized nitric bacteria.

FIG. 7b is a diagram of the relative ammonium degradation activity of biocatalysts on the basis of polyurethane and calcium alginate.

20 g of polyether polyol are used to produce the PCS prepolymers with a molecular weight of 2000 to 4000 and one Polyethylene oxide content <70% and 1.56 g TDI 80 (toluene diisocyanate, isomer ratio 2.4 / 2.6 is 80/20) mixed and kept in a sealed PE vessel at 75 ° C for 25 hours.  

The urethane prepolymer (PU-1) obtained in this way turns on finally dissolved in isopropanol. To a mixture of 40 g Prepolymer to 60 g of isopropanol is a with vigorous stirring Mixture of 4.1 g Na bisulfite in 68 g distilled water give. After 15 minutes the reaction is stopped by adjusting the pH value to 1.5 aborted using concentrated hydrochloric acid. The isopropanol is then stripped off at 100 mbar and 40 ° C. gene.

The water content of the product thus obtained is calculated and the sulfite content was determined titrimetrically. The pH of the If necessary, the product is corrected again to 1.5 so that it does not increase the viscosity of the PCS Prepolymer due to cross-linking reactions.

In order to investigate the influence of the capping on the toxicity of the isocyanate groups, the effect of tolylene diisocyanate and the TDI bisulfite adduct on Saccharomyces cerevisiae was checked. Fig. 1 shows very clearly that even at very low concentrations of free TDI there is only a residual activity of the microorganisms, while the influence of the TDI bisulfite adduct on S. cerevisiae is very small over a large concentration range.

The parameters, gel time and viscosity, which are particularly important for the processing of the PCS prepolymer, have been examined in detail. The results are shown in FIGS. 2 and 3. Fig. 2 shows that the PCS prepolymer cures very slowly at a pH <7 or <10 and is thus in a processable condition. In the pH range of 7-10, the crosslinking time drops rapidly and finds its minimum at a pH of something above 9, at which it is in the seconds range. The optimal pH range for the crosslinking reaction is approximately 8.5 to 9.5.

In Fig. 3, the dripping behavior of solutions having different concentrations PCS at certain pH values is illustrated. The dynamic viscosity as a function of the measurement duration serves as a measure of this. It was measured in a rotary viscometer (according to DIN) at D _rep = 704 s ^-1 . For the three measured PCS solutions, this means that a sufficiently long processing time is achieved. A sufficiently long processing time is a prerequisite for being able to influence the shape of the biocatalyst to be produced in a sufficient manner.

In order to produce spherical particles from polycarbamoylsulfonates, a solution is prepared which contains 10% PCS prepolymer, which was prepared according to Example 1, and 2% CaCl ₂ in water. The pH of this solution is adjusted to 6 and the cell mass is mixed in to produce the biocatalyst. The mixture is then dripped into a slightly alkaline Na alginate solution. Ca alginate shells immediately form around the drops and stabilize the shape. Due to the cross-linking reaction in the PCS core, which is triggered by the hydroxyl ions that diffuse through the Ca-alginate shell, the pH value drops slightly into the acidic range due to the release of sulfurous acid. The pH of the solution is kept in the slightly alkaline range by counter-titration with NaOH.

After 15 minutes, the beads obtained from the Na alginate Solution and put in another solution, which has a slightly alkaline pH. To cure the The core remains in this solution for 1 to 2 hours, at least in the first time it is re-titrated with NaOH, to keep the pH slightly alkaline.

The alginate shell is then covered with phosphate buffer or removed by stirring.

In Fig. 4, the procedure used in this example for the production of PCS beads by ionotropic gelation is shown graphically. First the PCS solution is mixed with a CaCl ₂ solution and the pH of the mixture is adjusted. The cell mass is then added and suspended in the solution. The decisive step in the production of PCS beads is done by dropping the suspension into a Na alginate solution with a pH of 8.5 using a syringe or a known immobilization apparatus. The simultaneous ionotropic shell formation and base-induced crosslinking of the core taking place is emphasized once again. Depending on the direction of diffusion of the Ca and hydroxyl ions, the Ca alginate shell is cross-linked from the inside to the outside, while the core cross-links from the outside to the inside. The last step in the process is the removal of the Ca-alginate shell, which takes place after the core has fully hardened.

In the reverse variant of the method, a Zellsus pension, which contains 5% PCS prepolymer and 2% Na alginate and is adjusted to a pH of 4.5, is added to a buffer solution with a pH of 8.5 2% CaCl ₂ is added, added dropwise. When dripped in, biocatalyst beads form which harden after one to two hours.

Another way to stabilize the shape of the Kataly sator is thermal. A 15% solution of PCS Prepolymer pre-set in water to approximately pH 6. This Lö solution is taken from a syringe into a 0.1 n heated to 40 ° C Buffer solution (phosphate buffer, pH 8.5) added dropwise. When on the polymer drops immediately turn milky white and keep their shape. After a while the pearls are gone the slightly basic pH is cured.

In a further process, the stabilizing shell is passed through Polyelectrolyte-polyelectrolyte crosslinking formed. Here is the Suspension adjusted to pH 6 and contains 10% PCS prepolymer and 1% polydimethyldiallylammonium chloride. This solution will dropped into a second solution which is adjusted to pH 8.5 and contains 1.5% cellulose sulfate. When dripping into the second  Solution immediately forms a thin shell while maintaining its shape out. The core is then cross-linked by diffusion Hydroxyl ions through this shell from the outside in. The born dete cover is so thin that it moves the substance in and out does not hinder the catalyst. Therefore, it does not need ent to be distant.

To compare the catalysts of the invention with to allow those that are known from the prior art The following are the results of three comparison subjects searches shown:

In FIGS. 5a and 5b is the activity of various Bioka talysatoren with immobilized cells of Saccharomyces cerevi siae shown in comparison to free cells. While the activity of biocatalysts based on alginate or PCS differ only slightly, biocatalysts based on uncapped polyurethane prepolymers (PU-1) show a significantly lower activity.

Fig. 5b clearly shows that biocatalysts from PCS beads are a competitive alternative to Ca-alginate biocatalyst beads in the activity of ethanol formation.

FIG. 6 shows a comparison of the nitrate degradation activity of Paracoccus denitrificans in matrices based on various NCO prepolymers and the PCS prepolymer. It can be seen very clearly that the PCS-based biocatalysts have a relatively high activity, while the biocatalysts based on other urethane prepolymers are practically inactive.

In FIGS. 7a and 7b, the ammonium degradation activity of mobilized in nitrifying bacteria (mainly Nitrobacter winograd sKyi and Nitrosomonas europeae) is shown. It turns out that in this case the activity of PCS-based biocatalysts is not achieved by other immobilizates.

However, it must be emphasized that in all examples the Free cell activity is greater than that of immobilized cells. It must be assumed that a certain pest delivery of the cells through the immobilization step for the acti loss of vitality is decisive. This loss of activity occurs even with the ionotropic, which is commonly referred to as very gentle Gel formation with Ca alginate. PCS-based biocatalysts usually show a comparable or even larger one Activity as biocatalysts based on Ca alginate. Only in the nitrate degradation activity could biocatalysts Based on PCS the activity of biocatalysts on Ca alginate do not reach. In contrast, biocatalysts show the basis of uncapped urethane prepolymers in all cases len a negligible or no activity.

Claims (22)

1. Biocatalyst with immobilized cells or enzymes, in which the cells or enzymes are immobilized in a matrix based on a polyurethane with blocked isocyanate groups, characterized in that the biocatalyst has a three-dimensional shape, which by introducing a first solution in a second solution can be produced in the second solution with immediate networking of the first solution. 2. Biocatalyst according to claim 1, characterized in that the matrix by a urethane prepolymer bisulfite ad duct (polycarbamoylsulfonate; PCS) is formed. 3. Biocatalyst according to claim 2, characterized in that the PCS matrix combines with another matrix is. 4. Process for the preparation of a catalyst according to An Proverb 1-3, characterized in that a Suspensi one that contains cells or enzymes through a central Nozzle is discharged, and the suspension with a capped polyurethane prepolymer and a first ver liquid containing network partner with the help of a  surrounding the central nozzle surrounding annular nozzle and is introduced into a second liquid, the contains a second cross-linking partner and a pH Value <7. 5. Process for the preparation of a catalyst of claims 1 to 3, characterized in that the Cells or enzymes in an aqueous solution of a ver capped polyurethane prepolymer with a first Vernet partner who is with a solution that is not in the solution Chen second network partner can respond to a pH value <7 that the so formed Suspension in a the second cross-linking partner liquid is introduced and that at or after Form a networked form of pH to <7 Crosslinking of the polyurethane is raised. 6. The method according to claim 4 or 5, characterized in that as a capped polyurethane prepolymer a urethane prep polymer bisulfite adduct (PCS) is used. 7. The method according to claim 4 to 6, characterized in that that the first crosslinking partner is an ionotropic, yellowish liquid and as a second cross-linking partner ionic crosslinking agent is used. 8. The method according to claim 4 to 7, characterized in that by using an ionic crosslinking agent as the first cross-linking partner and an ionotropic gel forming liquid as a second cross-linking partner the envelope surrounding the suspension is produced. 9. The method according to claim 4 to 6, characterized in that that ver as the first and second crosslinking partners be applied by wrapping a sheath around the suspension Form interfacial polymerization.   10. The method according to claim 8 or 9, characterized in that after the crosslinking of the polyurethane ent is removed. 11. The method according to claim 4 to 6, characterized in that that as the first and second networking partner Polyelektro lyte used to cover the suspension form through polyelectrolyte-polyelectrolyte crosslinking, the first crosslinking contained in the suspension partner is a quaternary ammonium salt. 12. A method for producing a biocatalyst according to An saying 1 or 2, characterized in that the cells or enzymes in the solution of the capped polyurethane pre polymers at a first temperature and pH Value <7 are suspended and that the suspension in a second liquid is introduced which has a pH <7 and has a second temperature higher than the first temperature and at which the polyurethane prepoly is no longer water-soluble and immediately keeps its shape fails. 13. Procedure according to. Claim 12, characterized in that the second liquid is formed by a buffer solution becomes. 14. The method according to any one of claims 4 to 13, characterized ge indicates that drops formed with the suspension who are dropped into the second liquid the. 15. The method according to any one of claims 4 to 14, characterized ge indicates that with the suspension a continuous Liquid jet is generated which flows into the second rivers liquidity is initiated.   16. The method according to any one of claims 4 to 14, characterized ge indicates that with the suspension drops with a central nozzle are formed when they emerge with the second liquid with the help of a central one Ring-shaped nozzle surrounding the nozzle. 17. The method according to any one of claims 4 to 16, characterized ge indicates that the pH of the suspension 6 turned on is posed. 18. The method according to claim 17, characterized in that the pH of the suspension is adjusted between 4 and 6 becomes. 19. The method according to any one of claims 4 to 18, characterized ge indicates that the pH <7 for crosslinking the Po lyurethans <8 is selected. 20. The method according to claim 19, characterized in that the pH <7 for crosslinking the polyurethane at about 9 is set. 21. The method according to any one of claims 6 to 20, characterized ge indicates that the PCS is based on a polyether polyols is formed. 22. The method according to any one of claims 6 to 21, characterized ge indicates that the polyurethane is based on a butt lyester polyol is formed.